Devices and Formulations for Detecting, Screening and Monitoring Levels of Certain Constituents in Bodily Fluids and Method

ABSTRACT

A device is disclosed for conducting a non-invasive analysis of a bodily fluid to determine the presence and level of a certain constituent carried by the bodily fluid. An indicator formulation of the device changes color in response to exposure to the constituent to provide a visible indication of the presence and level of the constituent carried by the bodily fluid. A carrier substrate of the device is constructed of a material having voids providing a high void volume within the substrate. The device is made by applying a chromagen to the carrier substrate to create a chromagen-laden carrier member. Then, a selected reagent having a particular constituent-specific formulation is applied to the chromagen-laden member. The selected reagent then combines with the chromagen thereby establishing the indicator formulation within the carrier substrate in place for reception of a sample of the bodily fluid.

This application is a continuation of U.S. patent application Ser. No.15/481,782, filed Apr. 7, 2017, which is a division of U.S. patentapplication Ser. No. 15/388,697, filed Dec. 22, 2016, now U.S. Pat. No.9,651,545, which is a division of U.S. patent application Ser. No.15/155,803, filed May 16, 2016, now U.S. Pat. No. 9,562,356, which is adivision of U.S. patent application Ser. No. 14/684,861, filed Apr. 13,2015, now U.S. Pat. No. 9,366,674, which is a continuation-in-part ofU.S. patent application Ser. No. 13/836,679, filed Mar. 15, 2013, nowU.S. Pat. No. 9,005,914, which is a continuation-in-part of U.S. patentapplication Ser. No. 13/606,299, filed Sep. 7, 2012, now U.S. Pat. No.8,431,386, which is a divisional application of U.S. application Ser.No. 13/278,306, filed Oct. 21, 2011, now U.S. Pat. No. 8,263,328, andfurther claims the benefit of U.S. Provisional Patent Application Ser.No. 61/455,528, filed Oct. 23, 2010, U.S. Provisional Patent ApplicationSer. No. 61/455,531, filed Oct. 23, 2010, U.S. Provisional PatentApplication Ser. No. 61/455,532, filed Oct. 23, 2010, and U.S.Provisional Patent Application Ser. No. 61/462,890, filed Feb. 9, 2011,the entire disclosures of which are incorporated herein by referencethereto.

The present invention relates generally to devices and formulations forreagents employed in such devices that enable detecting, screening andmonitoring levels of certain constituents in bodily fluids sampled fromhumans and animals, and pertains, more specifically, to the constructionand manufacture of such devices.

In two earlier U.S. Pat. Nos. 7,824,344 and 7,993,283, the substance ofwhich patents is incorporated herein by reference thereto, there isdisclosed methods and apparatus for conducting a non-invasive analysisof saliva. The present invention provides formulations and devices thatenable a user to employ a bodily fluid, such as saliva or another oralfluid, serum or plasma, utilizing devices that provide color changes toindicate the presence and level of a certain constituent in the bodilyfluid. Further, the present invention provides methods of constructionand manufacture that enable such devices to be made available forwidespread use for detecting, screening or monitoring the presence andlevel of any one of a plurality of certain constituents with increasedease and economy. As such, the present invention attains several objectsand advantages, some of which are summarized as follows: Providesdevices of simplified construction for widespread use in detecting,screening and monitoring the presence and level of any selected one of aplurality of certain constituents in bodily fluids; enables anexceptionally rapid response in a quick and easy non-invasive procedurefor determining the presence and level of a particular constituent in abodily fluid; provides for the economical manufacture and distributionof devices capable of detecting, screening and monitoring the presenceof certain constituents in bodily fluids; makes available a simplifiedvisual reading of a color change to determine the presence and level ofa certain constituent in a bodily fluid; provides an economical andreliable device for simplified use in detecting, screening or monitoringthe presence of a selected certain constituent in a bodily fluid;encourages widespread use to the benefit of a larger number of users whocan enjoy greater economy and convenience in reaching and maintaininghigher goals in healthcare.

The above objects and advantages are attained by the present invention,which may be described briefly as a method of making a device forconducting a non-invasive analysis of a bodily fluid to determine thepresence and the level of a certain constituent carried by the bodilyfluid, the device including an indicator formulation capable of changingcolor in response to exposure to the certain constituent to provide avisible indication of the presence and the level of the certainconstituent carried by the bodily fluid, the method comprising:providing a carrier substrate of a material having voids establishing ahigh void volume within the carrier substrate; applying a chromagenformulation to the carrier substrate to create a chromagen-laden carriermember; and subsequently applying to the chromagen-laden carrier membera selected reagent having a particular constituent-specific formulationto combine the selected reagent with the chromagen formulation appliedto the carrier substrate, thereby establishing the indicator formulationwithin the carrier substrate in place for reception of a sample of thebodily fluid later placed upon the carrier substrate; wherein thedifferent certain constituents are alanine aminotransferase (ALT) andaspartame aminotransferase (AST).

In addition, the present invention provides a device for conducting anon-invasive analysis of a bodily fluid to determine the presence andthe level of a certain constituent carried by the bodily fluid, thedevice including an indicator formulation capable of changing color inresponse to exposure to the certain constituent to provide a visibleindication of the presence and the level of the certain constituentcarried by the bodily fluid, the device comprising: a carrier substrateof a material having voids establishing a high void volume within thecarrier substrate; and an indicator formulation carried by the carriersubstrate, the indicator formulation consisting essentially of achromagen formulation and a constituent-specific formulation selectedfrom formulations responsive to levels of either one of the certainconstituents alanine aminotransferase (ALT) and aspartameaminotransferase (AST).

The present invention also provides a device for conducting anon-invasive analysis of a bodily fluid to determine the presence andthe level of a selected one of the low density and high density lipoidfractions of cholesterol carried by the bodily fluid, the deviceincluding an indicator formulation capable of changing color in responseto exposure to cholesterol to provide a visible indication of thepresence and the level of cholesterol carried by the bodily fluid, thedevice comprising: at least first and second test pads, each test padbeing comprised of a carrier substrate of a material having voidsestablishing a high void volume within the carrier substrate; anindicator formulation carried by the carrier substrate of each test pad,the indicator formulation consisting essentially of a chromagenformulation and a cholesterol-specific formulation responsive to thepresence and level of cholesterol; a treatment pad juxtaposed with thesecond test pad; and a lipoid precipitation agent carried by thetreatment pad, the lipoid precipitation agent being specific to theprecipitation of one of the low density and high density lipoidfractions of cholesterol such that upon applying a sample of the bodilyfluid to the first test pad a visible indication will be provided of thepresence and level of cholesterol carried by the bodily fluid, and uponapplying a further sample of the bodily fluid to the treatment pad, thefurther sample will pass through the treatment pad wherein the one ofthe low density and high density lipoid fractions will be removed priorto the further sample entering the second test pad to provide a visibleindication of the presence and level of the other of the low density andhigh density lipoid fractions of cholesterol in the bodily fluid,thereby enabling a determination of the presence and level of theselected one of the low density and high density lipoid fractionspresent in the bodily fluid.

In addition, the present invention provides a method for conducting anon-invasive analysis of a bodily fluid to determine the presence andthe level of a selected one of the low density and high density lipoidfractions of cholesterol carried by the bodily fluid, the methodincluding the provision of an indicator formulation capable of changingcolor in response to exposure to cholesterol to provide a visibleindication of the presence and the level of cholesterol carried by thebodily fluid, the method comprising: providing at least first and secondtest pads, each test pad being comprised of a carrier substrate of amaterial having voids establishing a high void volume within the carriersubstrate; including the indicator formulation in the carrier substrateof each test pad, the indicator formulation consisting essentially of achromagen formulation and a cholesterol-specific formulation responsiveto the presence and level of cholesterol; applying a sample of thebodily fluid to the first test pad; juxtaposing a treatment pad with thesecond test pad; providing a lipoid precipitation agent carried by thetreatment pad, the lipoid precipitation agent being specific to theprecipitation of one of the low density and high density lipoidfractions of cholesterol; applying the sample to the first test pad toderive a visible indication of the presence and level of totalcholesterol carried by the bodily fluid; applying a further sample tothe treatment pad wherein the one of the low density and high densitylipoid fractions will be removed; conducting the further sample from thetreatment pad to the second test pad to derive a visible indication ofthe presence and level of the other of the low density and high densitylipoid fractions of cholesterol in the bodily fluid, thereby enabling adetermination of the presence and level of the selected one of the lowdensity and high density lipoid fractions present in the bodily fluid.

Further, the present invention provides a method of screening fornon-alcoholic fatty liver disease (NAFLD) in a patient, said methodcomprising the steps of: (a) obtaining a biological sample from apatient; (b) providing a plurality of detector devices, wherein eachdetector device comprises at least one detection reagent whichspecifically binds to an analyte, wherein the detection reagentspecifically binds an analyte selected from the group consisting ofglucose, cholesterol, alanine aminotransferase (ALT), and aspartateaminotransferase (AST), wherein the binding of detection reagent toanalyte provides a visual indication of the presence and level ofanalyte in the sample, (c) applying the sample to the at least onedetector device, and (d) ascertaining the presence and level of saidanalyte in said sample.

Still further, the present invention provides a method of screening fornon-alcoholic fatty liver disease (NAFLD) in a patient, said methodcomprising the steps of: (a) obtaining a biological sample from apatient; (b) providing a detector device, wherein the detector devicecomprises a plurality of detection reagents which specifically binds toan analyte selected from the group consisting of glucose, cholesterol,alanine aminotransferase (ALT), aspartate aminotransferase (AST), andcombinations thereof, wherein the binding of detection reagent toanalyte provides a visual indication of the presence and level ofanalyte in the sample, (c) applying the sample to the detector device,and (d) ascertaining the presence and level of analyte in said sample.

In addition, the present invention provides a method of screening forliver cancer NAFLD-associated malignancies in a patient, said methodcomprising the steps of: (a) obtaining a biological sample from apatient; (b) providing a plurality of detector devices, wherein eachdetector device comprises a plurality of detection reagents whichspecifically binds to an analyte, selected from the group consisting ofglucose, cholesterol, alanine aminotransferase (ALT), aspartateaminotransferase (AST), and combinations thereof, wherein the binding ofdetection reagent to analyte provides a visual indication of thepresence and level of analyte in the sample, (c) applying the sample toat least one of the plurality of detector devices, and (d) ascertainingthe presence and level of analyte in said sample, wherein the screeningis positive for NAFLD-associated malignancies if elevated levels of atleast three of the analytes selected from the group consisting ofglucose, cholesterol, alanine aminotransferase (ALT), and aspartateaminotransferase (AST) relative to a predetermined reference rangeand/or control sample analyte are detected.

Further, the present invention provides a method of screening forNAFLD-associated malignancies in a patient, said method comprising thesteps of: (a) obtaining a biological sample from a patient; (b)providing a detector device, wherein the detector device comprises aplurality of detection reagents which specifically binds to an analyte,selected from the group consisting of glucose, cholesterol, alanineaminotransferase (ALT), aspartate aminotransferase (AST), andcombinations thereof, wherein the binding of detection reagent toanalyte provides a visual indication of the presence and level ofanalyte in the sample, (c) applying the sample to the detector device,and (d) ascertaining the presence and level of analyte in said sample,wherein the screening is positive for NAFLD-associated malignancies ifelevated levels of at least three of the analytes selected from thegroup consisting of glucose, cholesterol, alanine aminotransferase(ALT), and aspartate aminotransferase (AST) relative to a predeterminedreference range and/or control sample analyte are detected.

Still further, the present invention provides a method of screening forlevel of low density lipoid fractions of cholesterol in a patient, saidmethod comprising the steps of: (a) obtaining a biological sample from apatient; (b) providing a device for conducting a non-invasive analysisof a bodily fluid to determine the presence and the level of low densitylipoid factions of cholesterol carried by the bodily fluid, the deviceincluding an indicator formulation capable of changing color in responseto exposure to cholesterol to provide a visible indication of thepresence and the level of cholesterol carried by the bodily fluid, thedevice comprising: at least first and second test pads, each test padbeing comprised of a carrier substrate of a material having voidsestablishing a high void volume within the carrier substrate; anindicator formulation carried by the carrier substrate of each test pad,the indicator formulation consisting essentially of a chromagenformulation and a cholesterol-specific formulation responsive to thepresence and level of cholesterol; a treatment pad juxtaposed with thesecond test pad; and a lipoid precipitation agent carried by thetreatment pad, the lipoid precipitation agent being specific to theprecipitation of the low density lipoid factions of cholesterol; (c)applying a sample of the bodily fluid to the first test pad a visibleindication will be provided of the presence and level of cholesterolcarried by the bodily fluid, and upon applying a further sample of thebodily fluid to the treatment pad, the further sample will pass throughthe treatment pad wherein the low density lipoid factions will beremoved prior to the further sample entering the second test pad; and(d) ascertaining the presence and level of analyte in said sample,wherein the to provide a visible indication of the presence and level ofhigh density lipoid factions of cholesterol in the bodily fluid, therebyenabling a calculation to determine the presence and level of lowdensity lipoid factions present in the bodily fluid.

Additionally, the present invention provides a method of screening thepresence and the level of low density lipoid fractions of cholesterol ina patient, said method comprising the steps of: (a) obtaining abiological sample from a patient; (b) providing a device for conductinga non-invasive analysis of a bodily fluid to determine the presence andthe level of low density lipoid fractions of cholesterol carried by thebodily fluid, the device including an indicator formulation capable ofchanging color in response to exposure to cholesterol to provide avisible indication of the presence and the level of cholesterol carriedby the bodily fluid, the device comprising: at least first and secondtest pads, each test pad being comprised of a carrier substrate of amaterial having voids establishing a high void volume within the carriersubstrate; an indicator formulation carried by the carrier substrate ofeach test pad, the indicator formulation consisting essentially of achromagen formulation and a cholesterol-specific formulation responsiveto the presence and level of cholesterol; a treatment pad juxtaposedwith the second test pad; and a lipoid precipitation agent carried bythe treatment pad, the lipoid precipitation agent being specific to theprecipitation of the low density lipoid factions of cholesterol; (c)applying a sample of the bodily fluid to the first test pad a visibleindication will be provided of the presence and level of cholesterolcarried by the bodily fluid, and upon applying a further sample of thebodily fluid to the treatment pad, the further sample will pass throughthe treatment pad wherein the low density lipoid factions will beremoved prior to the further sample entering the second test pad toprovide a visible indication of the presence and level of high densitylipoid factions of cholesterol in the bodily fluid, thereby enabling acalculation to determine the presence and level of low density lipoidfactions present in the bodily fluid; and (d) ascertaining the presenceof analyte in said sample, wherein the to provide a visible indicationof the presence and level of high density lipoid factions of cholesterolin the bodily fluid, thereby enabling a calculation to determine thepresence and level of low density lipoid factions present in the bodilyfluid.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the term “patient” refers to an animal,preferably a mammal such as a non-primate (e.g., cows, pigs, horses,cats, dogs, rats etc.) and a primate (e.g., monkey and human), and mostpreferably a human. In some embodiments, the subject is a non-humananimal such as a farm animal (e.g., a horse, pig, or cow) or a pet(e.g., a dog or cat). In a specific embodiment, the subject is anelderly human. In another embodiment, the subject is a human adult. Inanother embodiment, the subject is a human child. In yet anotherembodiment, the subject is a human infant. Further, as used herein, theterm “predetermined reference range” refers to a reference range for theparticular patient, subject, or a population of subjects. Eachlaboratory may establish its own reference range for each particularassay, or a standard reference range for each assay may be madeavailable and used locally, regionally, nationally, or worldwide or maybe patient-specific. In one specific embodiment, the term refers to areference range for the amount of e.g., glucose, cholesterol, alanineaminotransferase (ALT), and/or aspartate aminotransferase (AST) in apatient or a specimen from a patient. In another specific embodiment,the term refers to a reference range for the amount of e.g., glucose,cholesterol,alanine aminotransferase (ALT), and/or aspartateaminotransferase (AST) in a patient or a specimen from a patient. Theassay may be run simultaneously with a second control assay wherein thecontrol sample does not contain elevated levels of analyte. The assaymay be run simultaneously with a control set of analyte standards togenerate a standard curve from which sample presence and levels ofanalyte can be quantitated.

The invention will be understood more fully, while still further objectsand advantages will become apparent, in the following detaileddescription of preferred embodiments of the invention illustrated in theaccompanying drawing, in which:

FIG. 1 is a pictorial view of a device constructed in accordance withthe present invention;

FIG. 2 is an enlarged, somewhat diagrammatic, cross-sectional view takenalong line 2-2 of FIG. 1;

FIG. 3 is a flow diagram illustrating a method of the present invention;

FIG. 4 is a plan view of another device constructed in accordance withthe present invention;

FIG. 5 is a pictorial view showing use of the device of FIG. 4;

FIG. 6 is a pictorial view showing the use of still another deviceconstructed in accordance with the present invention;

FIG. 7 is a pictorial view showing the use of yet another deviceconstructed in accordance with the present invention;

FIG. 8 is a fragmentary pictorial view of another device constructed inaccordance with the present invention;

FIG. 9 is an enlarged fragmentary cross-sectional view taken along line9-9 of FIG. 8;

FIG. 10 is a fragmentary pictorial view of yet another deviceconstructed in accordance with the present invention; and

FIG. 11 is a partially diagrammatic and enlarged fragmentarycross-sectional view taken along line 11-11 of FIG. 10.

Referring now to the drawings, and especially to FIGS. 1 and 2 thereof,a device constructed in accordance with the present invention is shownin the form of a dry-reagent device 20 and is seen to include a carriersubstrate in the form of a pad 22 of a material having voids 24establishing a high void volume within the pad 22. An indicatorformulation is carried by the pad 22 and is illustrated at 30 in theform of a layer 32 carried by fibers 34 of the material, injuxtaposition with voids 24 within the pad 22. Indicator formulation 30consists essentially of a chromagen formulation and a reagent having aconstituent-specific formulation selected from formulations responsiveto one of a plurality of constituents, as will be set forth in greaterdetail below. Suffice it to say at this juncture that the reagent havinga constituent-specific formulation and the chromagen formulation arecombined within the pad 22 such that the indicator formulation 30 iscapable of changing color in response to exposure to the certainconstituent to provide a visible indication on the pad 22 of thepresence and the level of the certain constituent carried by a sample ofa bodily fluid applied to the pad 22.

Thus, upon applying a sample of a bodily fluid, such as a saliva sample,to the pad 22, placed at a target area 36 of the device 20, theoccurrence of a visible color change will provide at least a qualitativeindication of the presence of the particular specific constituent towhich the constituent-specific formulation will react. An absence of anyvisible color change will indicate that the specific constituent is notpresent in any significant amount in the saliva sample.

The preferred material for pad 22 is a non-woven fibrous material whichprovides the requisite high void volume. The high void volume providespad 22 with the ability to absorb rapidly the sample of bodily fluidapplied to the target area 36, to enable rapid interaction of the samplewith the indicator formulation 30, and to maximize exposure of theinteracting sample and indicator formulation to ambient air forpromoting a quick response through accelerating a reaction between thecertain constituent carried by the sample and the indicator formulation.Non-woven synthetic polymeric materials are available commercially, onesuch material being a non-woven polyester fibrous material. Suitableglass-fiber non-woven fibrous materials and cellulose non-woven fibrousmaterials also are available commercially in forms suitable for use inthe construction of pad 22. The preferred materials are chosen toprovide pad 22 with a void volume within a range of about eight totwelve percent of the total volume of the material.

Device 20 is constructed in several different variations such that onevariation is available to provide a visible color change as anindication of at least the presence of a corresponding one of severalcertain constituents, namely, ethanol, uric acid, galactose, glucose,cholesterol, alanine aminotransferase (ALT) and aspartateaminotransferase (AST) and, preferably, the level of the certainconstituent, in the sample of bodily fluid applied to the target area 36of the pad 22. Each variation requires that the pad 22 carry aformulation specific to the constituent to be detected, as a componentof the indicator formulation 30; however, the chromagen formulationremains unchanged among the different variations of the pad 22 so thatthe same chromagen formulation can serve in every variation of the pad22. Accordingly, the manufacture and distribution of the devices 20 issimplified and rendered more economical, as will be described below.

Turning now to FIG. 3, as well as to FIGS. 1 and 2, pad 22 of device 20is manufactured by first applying to the material of pad 22 thechromagen formulation, as seen in step 40, to create a chromagen-ladencarrier member in the form of pad 22 with the chromagen formulationplaced in juxtaposition with voids 24 of the pad 22. Subsequently, aselected reagent having a particular constituent-specific formulation isapplied to the chromagen-laden carrier member, as indicated at step 42,to combine the selected reagent with the chromagen formulation appliedearlier to the 20 material of pad 22, thereby establishing the indicatorformulation 30 within the completed pad 22. Pad 22 is placed at targetarea 36 of device 20 for reception of the sample of bodily fluid uponthe pad 22. Since the chromagen formulation remains the same for allvariations of the device 20, economies are realized in the manufacturingprocess which requires only one component common to all variations andonly one station for the application of that common component; however,further economy and convenience are accomplished by the ability to storethe intermediate product, that is, the material of pad 22 with theapplied chromagen formulation, is stored, as seen in step 44.Subsequently, any selected one of the constituent-specific formulationsis applied, at a later time, in accordance with the requirement for anynumber of a particular device or particular devices, the completedcarrier member with the indicator formulation being available at step46. The ability to have on hand a supply of the basic chromagen-ladencarrier member for subsequent combination with a selectedconstituent-specific formulation, as opposed to immediately creating aninventory 10 of completed carrier members, as indicated by procedure 48,reduces the necessity for maintaining on-hand a large inventory of everyvariety of completed device 20 while increasing the flexibility andturn-around time of filling the demand for any number of devices 20 inany one of the different varieties.

With respect to the varieties identified above, a preferred commonchromagen formulation consists essentially of the following components,in an example prepared as follows: Approximately equal volumes of about0.05 to 0.5 M MBTH in distilled water are mixed with about 0.05 to 0.5 MDMAB in ethanol. An alternate formulation consists essentially ofapproximately 500 to 1,000 mg DAOS mixed with 100 ml ethanol. Themixture is impregnated into the material of the carrier member and theimpregnated material subsequently is dried, leaving the material withthe chromagen formulation coated upon the fibers of the material. Otherchromagen formulations compatible with the present reactions will becomeapparent to those persons of ordinary skill in the art.

With respect to each of the varieties identified above, the followingconstituent-specific formulations are effective, and an example of thepreparation of each is set forth below: For the determination of thepresence and level of ethanol as the certain constituent in a bodilyfluid, a constituent-specific formulation consists essentially of thefollowing components, in an example prepared as follows: Mix togetherapproximately equal amounts of about 1% to 20% 5 PVP K30 in distilledwater, about 0.5% to 5% ethoxylated surfactant in distilled water andabout 0.05 to 0.5 M phosphate buffer at pH 8.5 together with one-halfthe same amount of alcohol oxidase with an activity of approximately 400U/ml and one-quarter the same amount of peroxidase with an activity ofapproximately 200 U/mg. The prepared constituent-specific formulationthen is impregnated into the material previously impregnated with thechromagen formulation to complete 10 a pad 22 having an indicatorformulation 30 responsive to the presence and level of ethanol in anapplied sample of a bodily fluid.

For the determination of the presence and level of uric acid as thecertain constituent in a bodily fluid, a constituent-specificformulation consists essentially of the following components, in anexample prepared as follows: In approximately one-hundred ml of 0.05 to0.5 M phosphate buffered saline at pH 6.4, mix together approximatelyten mg of uricase, fifteen mg of ascorbate oxidase and about six mg ofperoxidase with an activity of approximately 200 U/mg. The preparedconstituent-specific formulation then is impregnated into the materialpreviously impregnated with the chromagen formulation to complete a pad22 having an indicator formulation 30 responsive to the presence andlevel of uric acid in an applied sample of a bodily fluid.

For the determination of the presence and level of galactose as thecertain constituent in a bodily fluid, a constituent-specificformulation consists essentially of the following components, in anexample prepared as follows: Mix together approximately five ml each ofabout 0.05 to 0.5 M phosphate buffer at pH 7.0, peroxidase with anactivity of about 200 U/mg, and ethanol (95%) together with abouttwenty-five ml of 10% polyvinyl alcohol in distilled water and 4200units of galactose oxidase. The prepared constituent-specificformulation then is impregnated into the material previously impregnatedwith the chromagen formulation to complete a pad 22 having an indicatorformulation 30 responsive to the presence and level of galactose in anapplied sample of a bodily fluid.

For the determination of the presence and level of glucose as thecertain constituent in a bodily fluid, a constituent-specificformulation consists essentially of the following components, in anexample prepared as follows: Dissolve approximately equal amounts of theenzymes glucose oxidase with an activity of approximately 200 U/mg andperoxidase with an activity of approximately 200 U/mg in distilled waterin the presence of approximately equal amounts of 0.05 to 0.5 M HEPES, ablend of surface active agents within the range of about 0.1% to 10%each, and a stabilizer, the preferred stabilizer being a PVP/copolymercomplex in which the copolymer is methylvinylether/maleic anhydride,available commercially under the trademark GANTREZO, the complex beingprepared from 5% PVP K30 in distilled water and 5% GANTREZO AN 139 at aPh of about 7.5. The prepared constituent-specific formulation then isimpregnated into the material previously impregnated with the chromagenformulation to complete a pad 22 having an indicator formulation 30responsive to the presence and level of glucose in an applied sample ofa bodily fluid.

For the determination of the presence and level of alanineaminotransferase (ALT) as the certain constituent in a bodily fluid, aconstituent-specific formulation consists essentially of the followingcomponents, mixed together in the indicated proportions: About 100 mg ofL-alanine, about 2200 units of pyruvate oxidase, about 560 mg of4-aminoantipyrine, about 10,000 units of peroxidase, about 12,000 unitsof ascorbate oxidase, about 400 mg of thiamine pyrophosphate, about 640mg of magnesium chloride and about 100 uL of 10% 0N870, all mixed withabout 100 ml of distilled water.

For the determination of the presence and level of aspartateaminotransferase (AST) as the certain constituent in a bodily fluid, aconstituent-specific formulation consists essentially of the followingcomponents, mixed together in the indicated proportions: About 200 mg ofsodium-L aspartate, about 16 mg of a-ketoglutaric acid, about 1060 mg ofoxalacetic acid decarboxylase, about 2200 units of pyruvate oxidase,about 640 mg of 4-aminoantipyrine, about 10,000 units of peroxidase,about 12,000 units of ascorbate oxidase, about 3.8 mg ofthiaminepyrophosphate, about 106.4 mg of magnesium chloride and about 100 uL of10% 0N870, all mixed with about 100 ml of distilled water.

For the determination of the presence and level of cholesterol as thecertain constituent in a bodily fluid, a constituent-specificformulation consists essentially of the following components, in anexample prepared as follows: Dissolve approximately equal amounts of theenzymes cholesterol esterase with an activity of approximately 180U/mgand peroxidase with an activity of approximately 200 U/mg and twice asmuch cholesterol oxidase with an activity of approximately 47 U/mg) indistilled water in the presence of approximately equal amounts of 0.05to 0.5 M HEPES, a blend of surface active agents within the range ofabout 0.1% to 10% each and a stabilizer, the preferred stabilizer beinga PVP/copolymer complex in which the copolymer ismethylvinylether/maleic anhydride, available commercially under thetrademark GANTREZO, the complex being prepared from 5% PVP K30 indistilled water and 5% GANTREZO AN 139 at a pH of about 7.5. Theprepared constituent-specific formulation then is impregnated into thematerial previously impregnated with the chromagen formulation tocomplete a pad 22 having an indicator formulation responsive to thepresence and level of cholesterol in an applied sample of a bodilyfluid.

Where it is desired further to determine the levels of both high densitylipoids (HDL) and low density lipoids (LDL), in addition to providing aquantitative numeric value for total lipoids (cholesterol), the presentinvention provides the ability to employ dry reagent techniques, asdisclosed above, lateral flow techniques, as disclosed in U.S. Pat. No.8,889,427, or a combination of both techniques to achieve that end.

Thus, for example, with reference to FIGS. 8 and 9, another deviceconstructed in accordance with the present invention is shown in theform of a dry-reagent device 220 that includes a target area 236 havingadjacent independent sections 240 and 242 placed for reception of asample of bodily fluid, such as a saliva sample. Section 240 includes atest pad 244 constructed as described above in connection with pad 22and carrying the constituent-specific formulation for the detection ofthepresence and level of cholesterol. A second test pad 246 is locatedremote from first test pad 244 and also is constructed as describedabove in connection with pad 22, carrying the constituent specificformulation for detecting cholesterol.

Section 242 includes a treatment pad 250 that carries a lipoidprecipitation agent, such as an organic acid, specific to theprecipitation of the LDL fraction of cholesterol. Thus, upon applying tothe target area 236 a sample of a bodily fluid, such as a saliva sample,a first portion of the sample will enter directly into test pad 244,while a second portion of the sample will enter treatment pad 250 toproceed through treatment pad 250 to test pad 246. A filter member 252is interposed between treatment pad 250 and test pad 246 so that passageof liquid precipitation agent from treatment pad 250 into test pad 246is precluded. Further, a barrier 254 isolates section 240 from section242, thereby assuring that liquid precipitation agent cannot migratefrom treatment pad 250 into test pad 244.

Accordingly, upon introducing a sample of bodily fluid, such as a salivasample, to the target area 236, a portion of the sample will enter testpad 244 and a color change indicative of the total level of cholesterolpresent in the sample portion will appear at a surface 256 of test pad244, while another portion of the sample will enter treatment pad 250,will pass through treatment pad 250, and will enter test pad 246, sothat a color change indicative of the level of the HDL fraction ofcholesterol present in the sample portion will appear at a surface 258of the test pad 246, the LDL fraction having been removed at thetreatment pad 250. These results for the levels of total cholesterol andof the HDL fraction then are used in connection with a standardalgorithm to calculate the level of the LDL fraction present in thebodily fluid. Alternately, the level of the LDL fraction can bedetermined directly by removing the HDL fraction with an appropriatelipoid precipitation agent in the treatment pad 250 so that the colorchange at test pad 246 will be indicative of the level of the LDLfraction of cholesterol present in the sample portion. Should it furtherbe desired to determine the level of very low density lipoids (VLDL) inthe bodily fluid, similar separation, indication and calculationtechniques can be employed.

Referring now to FIGS. 10 and 11, another device constructed inaccordance with the present invention is shown in the foi n of a lateralflow device 320, as described more fully in the aforesaid U.S. Pat. No.8,889,427, the disclosure of which is incorporated herein by referencethereto. Device 320 includes a test strip 322 having a target area 324placed for reception of a sample of bodily fluid, such as a salivasample. Test strip 322 is constructed of a porous material that enablesremoval of a first portion of the sample laterally, as illustrateddiagrammatically at 326 in FIG. 11, to a first test pad 330 whichcarries a constituent-specific formulation for the detection of thepresence and level of cholesterol. A second portion of the sample ismoved laterally, as illustrated diagrammatically at 336, toward a secondtest pad 340 placed adjacent first test pad 330, the second test pad 340being isolated from first test pad 330 by a barrier 342. Test pad 340likewise carries the constituent specific formulation for detectingcholesterol. A treatment pad 350 is interposed between the target area324 and the second test pad 340, in position to receive the secondportion of the sample from the target area 324. Treatment pad 350carries a lipoid precipitation agent, such as an organic acid, specificto the precipitation of the LDL fraction of cholesterol. The secondportion of the sample, now treated within treatment pad 350 to removethe LDL fraction, moves laterally, as illustrated at 352, and passesthrough a filter member 354 that precludes passage of the liquidprecipitation agent from treatment pad 350 into test pad 340.Accordingly, as further illustrated diagrammatically in FIG. 11, uponintroducing a sample of bodily fluid, such as a saliva sample, to thetarget area 324, a color change at test pad 330 indicative of the totallevel of cholesterol present in the sample will be read at 360, while acolor change indicative of the level of the HDL fraction remaining afterremoval of the LDL fraction at treatment pad 350 will be read at 362.These results for the levels of total cholesterol and of the HDLfraction then are used in connection with a standard algorithm tocalculate, at 364, the level of the LDL fraction present in the bodilyfluid, which level is displayed at 366. Alternately, the level of theLDL fraction can be determined directly by removing the HDL fractionwith an appropriate lipoid precipitation agent in the treatment pad 350so that the color change at test pad 340 will be indicative of the levelof the LDL fraction of cholesterol present in the sample portion.

It will be apparent that the placement of test pads 244 and 246, as wellas test pads 330 and 340, can be in parallel, or in series, or in anyfeasible relationship adjacent to one another enabling the flow ofsample material into corresponding test pads. Thus, the test pads can beon the same or on opposite sides of a test strip. Any reading of theresults of a test can be made from the top side or from the bottom sideof the test strip, or from both sides.

The importance of screening for the presence and level of each of thevarious constituents identified above is well-known. Of particularinterest, however, is the ability to determine the existence of specificdiseases, based upon the presence of certain combinations of thesedetected constituents. For example, a patient exhibiting elevated levelsof ALT or AST presents a possibility of the presence of non-alcoholicfatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), adisease growing to epidemic proportions globally. Accordingly, uponutilization of the present invention to screen for the determination ofthe presence and level of certain of the herein enumerated constituents,a patient having signs of elevated levels of cholesterol AND elevatedlevels of glucose, accompanied by elevated levels of ALT AND elevatedlevels of AST, the patient is very likely to have NAFLD or NASH. Apatient having signs of elevated levels of cholesterol OR elevatedlevels of glucose, accompanied by elevated levels of ALT AND elevatedlevels of AST is likely to have NAFLD or NASH. A patient having signs ofelevated levels of cholesterol AND elevated levels of glucose,accompanied by elevated levels of ALT OR elevated levels of AST, thepatient is likely to have NAFLD or NASH. Thus, in summary, a patienthaving elevations of three or four of the markers cholesterol, glucose,ALT and AST is likely to have NAFLD or NASH, and would require furthermedical investigation. When screening for NAFLD or NASH, in accordancewith the present invention, a biological sample can be a member selectedfrom the group consisting of whole blood, serum, plasma, cerebrospinalfluid, saliva, urine, spinal fluid, synovial fluid, amniotic fluid andcranial fluid, and lymphocyte or cell culture supernatants. Inconnection with the formulations set forth above, the detection reagentcan be selected from the group consisting of chromagens; antigens;haptens; monoclonal and polyclonal antibodies; natural and syntheticmono-, oligo- and polysaccharides; lectins; avidin and streptavidin;biotin; growth factors; hormones; receptor molecules; and combinationsthereof.

In addition, it is noted that current indications are that NAFLD cancause primary liver cancer, in particular hepatocellular carcinoma(HCC), so reliable screening protocols for the presence of NAFLD becomeof even greater importance for determining populations requiring moreaggressive screening for liver cancer. With respect to HCC, anotoriously chemo-resistant tumor, it is likely that further newtreatments will be necessitated by such an explosion in NAFLD associatedmalignancies. Addressing this possibility, glucocorticoid-receptor (GR)often is expressed in the nuclei of HCC, indicating that the tumor islikely responsive to the chemo sensitizing effects of Org34517, as hasalready been demonstrated in xenograft models of human, GR-expressingovarian and breast carcinomas, as set forth in U.S. Pat. No. 8,658,128.

In the embodiment of the invention illustrated in FIG. 4, a disk-shapeddevice 100 includes a pad 110 constructed of the material previouslydescribed in connection with pad 22 of device 20. Pad 110 provides atarget area 112 which, in device 110, is substantially surrounded by anintegrated color gauge 114 having patches 116 of different colors thatcan be matched visually with a color change at the target area 112. Asin the devices disclosed in the aforesaid patents, U.S. Pat. Nos.7,824,344 and 7,993,283, alpha-numeric characters may be displayed injuxtaposition with the patches 116 for a direct reading of the leveldetected. In this manner, device 100 provides a simple semiqualitative/semi-quantitative measure of the presence and level of aparticular certain constituent carried by a sample of bodily fluidapplied to the target area 112. The semi-qualitative/semi quantitativeindication, while more comprehensive than the generally qualitativeindication provided by device 20 described above, is convenient, asshown in FIG. 5 where the device 100 is placed readily within the palm118 of a user's hand, but not as comprehensive as the indicationprovided by other embodiments of the invention, as set forth below.

In the embodiment shown in FIG. 6, a device 120 is constructed asdescribed in detail in the aforesaid patents, U.S. Pat. Nos. 7,824,344and 7,993,283, and is inserted into a reader 122 that employs analgorithm which converts a sensed color change into an accurate digitalreadout at a display 124, for a more accurate quantitative evaluation ofthe presence and level of a particular certain constituent carried by abodily fluid sample.

In the embodiment illustrated in FIG. 7, a device 130 carries a strip132 similar in construction to pad 22 described above, and arranged in acoil 134 held within the device 130. A sample of bodily fluid is appliedto the strip 132, at a target area registered with an access door 140through which the sample is passed to the strip 132. A color change issensed and an algorithm converts the sensed color change into anaccurate digital readout at a display 142. The used portion144 of thestrip 132 is advanced through a slot 146 and is torn off and discarded.

The embodiments illustrated in FIGS. 6 and 7 are conveniently availableto a user wishing to control and maintain weight levels. Thus, forexample, upon use of a device 120 or 130 in connection with monitoringthe level of glucose in a bodily fluid, such as saliva, the algorithmsprovided by these embodiments can convert the detected glucose levelinto glycemic control readily understood and employed by a user inconnection with a weight control regimen. In this manner, the user isprovided with information to refine glycemic control, enabling the userto adjust diet, supplement intake and exercise for the purpose ofglycemic control and weight control.

In another embodiment of the present invention, a dry-reagent device, alateral flow device, or another test device, preferably constructed inthe form of a dipstick, is incorporated into a kit suitable for hometesting. A screening test, as described above, would provideconvenience, privacy and eliminate the necessity and cost of visiting aphysician for a screening test, although the dipstick or other testdevice-based kit could also be used in a clinical setting. The dipstickor other test device-based kit would be similar to a home pregnancy kit,known to those of skill in the art, and could provide a color indicationof, or an increased risk for, e.g., NAFLD/NASH and the like describedherein, e.g., an analyte such as glucose, cholesterol, alanineaminotransferase (ALT), and aspartate aminotransferase (AST). Such atest device-based kit could be provided with a small plastic cup forcollecting and retaining the sample and for conducting the test. In onescenario, the dipstick or other test device could react to produce onecolor if a level of a first analyte, a different color if a level of,e.g., a second analyte is exceeded, and when both levels are exceeded,the two colors would combine to yield a third color easilydistinguishable from the others.

It will be seen that the present invention attains all of the objectsand advantages summarized above, namely: Provides devices of simplifiedconstruction for widespread use in detecting, screening and monitoringthe presence and level of any selected one of a plurality of certainconstituents in bodily fluids; enables an exceptionally rapid responsein a quick and easy non invasive procedure for determining the presenceand level of a particular constituent in a bodily fluid; provides forthe economical manufacture and distribution of devices capable ofdetecting, screening and monitoring the presence of certain constituentsin bodily fluids; makes available a simplified visual reading of a colorchange to determine the presence and level of a certain constituent in abodily fluid; provides an economical and reliable device for simplifieduse in detecting, screening or monitoring the presence of a selectedcertain constituent in a bodily fluid; encourages widespread use to thebenefit of a larger number of users who can enjoy greater economy andconvenience in reaching and maintaining higher goals in healthcare.

It is to be understood that the above detailed description of preferredembodiments of the invention is provided by way of example only. Variousdetails of design, construction and procedure may be modified withoutdeparting from the true spirit and scope of the invention, as set forthin the appended claims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A device for conductinga non-invasive analysis of a bodily fluid to determine the presence andthe level of a selected one of the low density and high density lipoidfractions of cholesterol carried by the bodily fluid, the deviceincluding an indicator formulation capable of changing color in responseto exposure to cholesterol to provide a visible indication of thepresence and the level of cholesterol carried by the bodily fluid, thedevice comprising: at least first and second test pads, each test padbeing comprised of a carrier substrate of a material having voidsestablishing a high void volume within the carrier substrate; anindicator formulation carried by the carrier substrate of each test pad,the indicator formulation consisting essentially of a chromagenformulation and a cholesterol-specific formulation responsive to thepresence and level of cholesterol; a treatment pad juxtaposed with thesecond test pad; and a lipoid precipitation agent carried by thetreatment pad, the lipoid precipitation agent being specific to theprecipitation of one of the low density and high density lipoidfractions of cholesterol such that upon applying a sample of the bodilyfluid to the first test pad a visible indication will be provided of thepresence and level of cholesterol carried by the bodily fluid, and uponapplying a further sample of the bodily fluid to the treatment pad, thefurther sample will pass through the treatment pad wherein the one ofthe low density and high density lipoid fractions will be removed priorto the further sample entering the second test pad to provide a visibleindication of the presence and level of the other of the low density andhigh density lipoid fractions of cholesterol in the bodily fluid,thereby enabling a determination of the presence and level of theselected one of the low density and high density lipoid fractionspresent in the bodily fluid.
 2. The device of claim 1 wherein theselected one of the low density and high density lipoid factions is thelow density lipoid factions, and the lipoid precipitation agent isspecific to the precipitation of low density lipoid fractions, therebyproviding a visible indication of the presence and level of high densitylipoid fractions of cholesterol in the bodily fluid and enabling acalculation to determine the presence and level of low density lipoidfractions present in the bodily fluid.
 3. The device of claim 2including: a target area for the reception of a single sample of thebodily fluid, the treatment pad being juxtaposed with the target areaand interposed between the target area and the second test pad; thetarget area being juxtaposed with the first test pad in directcommunication with the first test pad, and being juxtaposed with thetreatment pad in direct communication with the treatment pad, such thatupon applying the single sample to the target area, a first portion ofthe single sample will enter the first test pad to provide the visibleindication of the presence and level of cholesterol carried by thebodily fluid, while a second portion of the single sample will passthrough the treatment pad wherein the low density lipoid fractions willbe removed prior to the second portion of the single sample entering thesecond test pad to provide the visible indication of the presence andlevel of high density lipoid fractions of cholesterol in the bodilyfluid, thereby enabling the calculation to determine the presence andlevel of low density lipoid fractions present in the bodily fluid. 4.The device of claim 3 including a filter member interposed between thetreatment pad and the second test pad for precluding the passage oflipoid precipitation agent from the treatment pad to the second testpad.
 5. The device of claim 3 wherein the carrier substrate has a totalvolume and the high void volume is within a range of about eight totwelve percent of the total volume.